The present invention is directed to increased secretion of desired polypeptides from filamentous fungi. The invention discloses fusion nucleic acids, vectors, fusion polypeptides, and processes for obtaining the desired polypeptide.
Production of fusion polypeptides has been reported in a number of organisms, including E. coli, yeast, and filamentous fungi. For example, bovine chymosin and porcine pancreatic prophospholipase A2 have both been produced in A. niger or A. niger var. awamori as fusions to full-length GAI (U.S. Ser. No. 08/318,494; Ward et al., Bio/technology 8: 435-440,1990; Roberts et al.,Gene 122: 155-161, 1992). Human interleukin 6 (hIL6) has been produced in A. nidulans as a fusion to full-length A. niger GAI (Contreras et al., Biotechnology 9: 378-381, 1991). Hen egg white lysozyme (Jeenes et al., FEMS Microbiol. Lett. 107: 267-272, 1993) and human lactoferrin (Ward et al., Bio/technology 13: 498-503, 1995) have been produced in A. niger as fusions to residues 1-498 of glucoamylase and hIL6 has been produced in A. niger as a fusion to glucoamylase residues 1-514 (Broekhuijsen et al., J. Biotechnol. 31: 135-145, 1993). In some of the above experiments (Contreras et al., 1991; Broekhuijsen et al., 1993; Ward et al., 1995) a KEX2 protease recognition site (Lys, Arg) has been inserted between glucoamylase and the desired polypeptide to allow in vivo release of the desired polypeptide from the fusion protein as a result of the action of a native Aspergillus KEX2-like protease.
Additionally, bovine chymosin has been produced in A. niger as a fusion with full-length native alpha-amylase (Korman et al., Curr. Genet. 17: 203-212, 1990) and in A. oryzae as a fusion with truncated forms of A. oryzae glucoamylase (either residues 1-603 or 1-511; Tsuchiya et al., Biosci. Biotech. Biochem. 58: 895-899, 1994).
A small protein (epidermal growth hormone; 53 amino acids) has been produced in Aspergillus as a tandem fusion of three copies of the protein (U.S. Pat. No. 5,218,093). The trimer of EGF was secreted as a result of the inclusion of an N-terminal secretion signal sequence. However, the EGF molecules were not additionally fused to a protein efficiently secreted by filamentous fungi and no method for subsequent separation of monomeric EGF proteins was provided.
The glaA gene encodes glucoamylase which is highly expressed in many strains of Aspergillus niger and Aspergillus awamori. The promoter and secretion signal sequence of the gene have been used to express heterologous genes in Aspergillus including bovine chymosin in Aspergillus nidulans and A. awamori as previously described (Gwynne, D. et al. (1987) Bio/Technology 5, 713-719 and EPO Publication No. 0 215 594). In the latter experiments, a variety of constructs were made, incorporating prochymosin cDNA, either the glucoamylase or the chymosin secretion signal and, in one case, the first 11 codons of mature glucoamylase. Maximum yields of secreted chymosin obtained from A. awamori were below 15 mg/l in 50 ml shake flask cultures and were obtained using the chymosin signal sequence encoded by pGRG3. These previous studies indicated that integrated plasmid copy number did not correlate with chymosin yields, abundant polyadenylated chymosin mRNA was produced, and intracellular levels of chymosin were high in some transformants regardless of the source of secretion signal. It was inferred that transcription was not a limiting factor in chymosin production but that secretion may have been inefficient. It was also evident that the addition of a small amino terminal segment (11 amino acids) of glucoamylase to the propeptide of prochymosin did not prevent activation to mature chymosin. The amount of extracellular chymosin obtained with the first eleven codons of glucoamylase, however, was substantially less than that obtained when the glucoamylase signal was used alone. Subsequently, it was demonstrated that chymosin production could be greatly increased when a fusion protein consisting of full-length glucoamylase and prochymosin was produced (U.S. Ser. No. 08/318,494; Ward et al. Bio/technology 8: 435-440, 1990).
Aspergillus niger and Aspergillus niger var. awamori (A. awamori) glucoamylases have identical amino acid sequences. The glucoamylase is initially synthesized as preproglucoamylase. The pre and pro regions are removed during the secretion process so that mature glucoamylase is released to the external medium. Two forms of mature glucoamylase are recognized in culture supernatants: GAI is the full-length form (amino acid residues 1-616) and GAII is a natural proteolytic fragment comprising amino acid residues 1-512. GAI is known to fold as two separate domains joined by an extended linker region. The two domains are the 471 residue catalytic domain (amino acids 1-471) and the 108 residue starch binding domain (amino acids 509-616), the linker region being 36 residues in length (amino acids 472-508). GAII lacks the starch binding domain. These details of glucoamylase structure are reviewed by Libby et al. (Protein Engineering 7: 1109-1114, 1994) and are shown diagrammatically in FIG. 1.
Trichoderma reesei produces several cellulase enzymes, including cellobiohydrolase I (CBHI), which are folded into two separate domains (catalytic and binding domains) separated by an extended linker region. Foreign polypeptides have been secreted in T. reesei as fusions with the catalytic domain plus linker region of CBHI (Nyyssonen et al., Bio/technology 11: 591-595, 1993).
An object of the invention herein is to provide for the expression and secretion of desired polypeptides by and from filamentous fungi including fusion nucleic acids, expression vectors containing such fusion nucleic acids, transformed filamentous fungi, fusion polypeptides and processes for expressing and secreting high levels of such desired polypeptides.
In accordance with the above objects, the invention provides fusion nucleic acids encoding a fusion polypeptide comprising, from a 5xe2x80x2 end of the fusion nucleic acid, first, second, third and fourth nucleic acids. The first nucleic acid encodes a signal polypeptide functional as a secretory sequence in a first filamentous fungus. The second nucleic acid encodes a secreted polypeptide or functional portion normally secreted from a filamentous fungus. The third nucleic acid encodes a cleavable linker and the fourth nucleic acid comprises two or more nucleic acids each encoding desired polypeptides.
Further provided are fusion nucleic acids wherein the fourth nucleic acid further comprises at least one nucleic acid encoding a cleavable linker. The nucleic acids encoding said desired polypeptides are separated by the nucleic acid encoding the cleavable linker.
In another aspect, the invention provides a fusion nucleic acid encoding a fusion polypeptide comprising, from a 5xe2x80x2 end of the fusion nucleic acid, first, fifth, third and second nucleic acids. The fifth nucleic acid comprises at least one nucleic acid encoding a desired polypeptide.
In a further aspect the invention provides a fusion polypeptide comprising a first nucleic acid, a second nucleic acid, and an insertion nucleic acid. The insertion nucleic acid comprises a fifth nucleic acid flanked by third nucleic acids.
Also provided are expression vectors for transforming a host filamentous fungus comprising nucleic acids encoding regulatory sequences functionally recognized by said host filamentous fungus including promoter and transcription and translation initiation sequences operably linked to the 5xe2x80x2 end of the fusion nucleic acids described herein.
Although cleavage of the fusion polypeptide to release the desired polypeptide will often be useful, it is not necessary. In those cases in which the desired polypeptide retains its functionality when it is part of the fusion, polypeptide cleavage may not be required or desirable. For this reason, said third amino acid sequence comprising the cleavable linker may be considered optional.
Also provided are filamentous fungi containing a fusion nucleic acid described herein.
Additionally provided are fusion polypeptides comprising, from the amino-to caboxy-terminus, first, second, third and fourth amino acid sequences. The first amino acid sequence comprises a signal polypeptide functional as a secretory sequence in a filamentous fungus. The second amino acid sequence comprises a secreted polypeptide or functional portion normally secreted from a filamentous fungus. The third amino acid sequence comprises a cleavable linker and the fourth amino acid sequence comprises at least two desired polypeptides.
Further provided are fusion polypeptides comprising, from the amino-to caboxy-terminus, first, fifth, third and second amino acids. The fifth polypeptide comprises at least one desired polypeptide.
Also provided are fusion polypeptides comprising a first amino acid sequence, a second amino acid sequence, and an insertion amino acid sequence. The insertion amino acid sequence comprises a fifth amino acid sequence flanked by third amino acid sequences, and is inserted into the second amino acid sequence.
Additionally provided are processes for producing a desired polypeptide comprising transforming a host filamentous fungus with an expression vector containing a fusion nucleic acid described herein under conditions which permit expression of the fusion nucleic acid to cause the secretion of the desired polypeptide encoded by the fusion nucleic acid.